Bottom Line:
Using nuclear staining, the MTT assay and Western blotting analysis of the expression of cell-cycle markers, the proliferation rate of EMSCs on a fibrin matrix was found to be significantly higher than that of cells grown on a plastic surface but insignificantly lower than that of cells grown on fibronectin.Furthermore, the EMSCs grown on the fibrin matrix synthesized more neurotrophins compared with those grown on fibronectin or a plastic surface.These results demonstrated that fibrin not only promoted EMSC proliferation but also the differentiation of EMSCs into the SLCs.

Affiliation: Department of Histology and Embryology, School of Medicine, Jiangsu University, Zhenjiang, China.

ABSTRACTBecause Schwann cells perform the triple tasks of myelination, axon guidance and neurotrophin synthesis, they are candidates for cell transplantation that might cure some types of nervous-system degenerative diseases or injuries. However, Schwann cells are difficult to obtain. As another option, ectomesenchymal stem cells (EMSCs) can be easily harvested from the nasal respiratory mucosa. Whether fibrin, an important transplantation vehicle, can improve the differentiation of EMSCs into Schwann-like cells (SLCs) deserves further research. EMSCs were isolated from rat nasal respiratory mucosa and were purified using anti-CD133 magnetic cell sorting. The purified cells strongly expressed HNK-1, nestin, p75(NTR), S-100, and vimentin. Using nuclear staining, the MTT assay and Western blotting analysis of the expression of cell-cycle markers, the proliferation rate of EMSCs on a fibrin matrix was found to be significantly higher than that of cells grown on a plastic surface but insignificantly lower than that of cells grown on fibronectin. Additionally, the EMSCs grown on the fibrin matrix expressed myelination-related molecules, including myelin basic protein (MBP), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and galactocerebrosides (GalCer), more strongly than did those grown on fibronectin or a plastic surface. Furthermore, the EMSCs grown on the fibrin matrix synthesized more neurotrophins compared with those grown on fibronectin or a plastic surface. The expression level of integrin in EMSCs grown on fibrin was similar to that of cells grown on fibronectin but was higher than that of cells grown on a plastic surface. These results demonstrated that fibrin not only promoted EMSC proliferation but also the differentiation of EMSCs into the SLCs. Our findings suggested that fibrin has great promise as a cell transplantation vehicle for the treatment of some types of nervous system diseases or injuries.

f4-molcell-38-3-221: Proliferation of EMSCs grown on a plastic surface, fibronectin or the fibrin matrix. Nuclei of EMSCs grown on a plastic surface (A), fibronectin (B), or the fibrin matrix (C). (D) Cell density of EMSCs cultured for 12 days; (E) The results of the MTT assay showed the different rates of proliferation of EMSCs grown on the three substrates. (F) Representative Western blot of cell-cycle markers p21, p16, and PCNA, with β-actin serving as the loading control; (G) quantitative analysis of the Western blot; each bar represents the mean value ± SEM of measurements made in four independent replicates. Bar = 10 μm; *P < 0.05, **P < 0.01.

Mentions:
Although the EMSCs could be cultured for several months on a plastic surface by passaging them, it was still unclear whether these cells could survive on the fibrin matrix. Therefore, the EMSCs were cultured on the fibrin matrix for 12 days. As shown in Fig. 4C, Hoechst 33342-stained nuclei of the EMSCs were distributed over the entire fibrin matrix. Some cells wrapped around the fibrin filaments and formed a cylindrical structure (Fig. 4C). The density of nuclei observed on the fibrin matrix was significantly higher than that on a plastic surface but was insignificantly lower than that on fibronectin (Fig. 4D).

f4-molcell-38-3-221: Proliferation of EMSCs grown on a plastic surface, fibronectin or the fibrin matrix. Nuclei of EMSCs grown on a plastic surface (A), fibronectin (B), or the fibrin matrix (C). (D) Cell density of EMSCs cultured for 12 days; (E) The results of the MTT assay showed the different rates of proliferation of EMSCs grown on the three substrates. (F) Representative Western blot of cell-cycle markers p21, p16, and PCNA, with β-actin serving as the loading control; (G) quantitative analysis of the Western blot; each bar represents the mean value ± SEM of measurements made in four independent replicates. Bar = 10 μm; *P < 0.05, **P < 0.01.

Mentions:
Although the EMSCs could be cultured for several months on a plastic surface by passaging them, it was still unclear whether these cells could survive on the fibrin matrix. Therefore, the EMSCs were cultured on the fibrin matrix for 12 days. As shown in Fig. 4C, Hoechst 33342-stained nuclei of the EMSCs were distributed over the entire fibrin matrix. Some cells wrapped around the fibrin filaments and formed a cylindrical structure (Fig. 4C). The density of nuclei observed on the fibrin matrix was significantly higher than that on a plastic surface but was insignificantly lower than that on fibronectin (Fig. 4D).

Bottom Line:
Using nuclear staining, the MTT assay and Western blotting analysis of the expression of cell-cycle markers, the proliferation rate of EMSCs on a fibrin matrix was found to be significantly higher than that of cells grown on a plastic surface but insignificantly lower than that of cells grown on fibronectin.Furthermore, the EMSCs grown on the fibrin matrix synthesized more neurotrophins compared with those grown on fibronectin or a plastic surface.These results demonstrated that fibrin not only promoted EMSC proliferation but also the differentiation of EMSCs into the SLCs.

Affiliation:
Department of Histology and Embryology, School of Medicine, Jiangsu University, Zhenjiang, China.

ABSTRACTBecause Schwann cells perform the triple tasks of myelination, axon guidance and neurotrophin synthesis, they are candidates for cell transplantation that might cure some types of nervous-system degenerative diseases or injuries. However, Schwann cells are difficult to obtain. As another option, ectomesenchymal stem cells (EMSCs) can be easily harvested from the nasal respiratory mucosa. Whether fibrin, an important transplantation vehicle, can improve the differentiation of EMSCs into Schwann-like cells (SLCs) deserves further research. EMSCs were isolated from rat nasal respiratory mucosa and were purified using anti-CD133 magnetic cell sorting. The purified cells strongly expressed HNK-1, nestin, p75(NTR), S-100, and vimentin. Using nuclear staining, the MTT assay and Western blotting analysis of the expression of cell-cycle markers, the proliferation rate of EMSCs on a fibrin matrix was found to be significantly higher than that of cells grown on a plastic surface but insignificantly lower than that of cells grown on fibronectin. Additionally, the EMSCs grown on the fibrin matrix expressed myelination-related molecules, including myelin basic protein (MBP), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and galactocerebrosides (GalCer), more strongly than did those grown on fibronectin or a plastic surface. Furthermore, the EMSCs grown on the fibrin matrix synthesized more neurotrophins compared with those grown on fibronectin or a plastic surface. The expression level of integrin in EMSCs grown on fibrin was similar to that of cells grown on fibronectin but was higher than that of cells grown on a plastic surface. These results demonstrated that fibrin not only promoted EMSC proliferation but also the differentiation of EMSCs into the SLCs. Our findings suggested that fibrin has great promise as a cell transplantation vehicle for the treatment of some types of nervous system diseases or injuries.